The spindle checkpoint delays the progression of metaphase to anaphase until all chromosomes are properly attached to the mitotic spindle. A loss of the checkpoint function often results in genetic instability, which predisposes cells to malignant transformation. To understand the molecular basis of functional inactivation of this surveillance mechanism in human cancer development, the PI's laboratory has focused on the roles of two protein kinases, hBUB1 and hBUBR1 (42;53;81), mutations of which are detected in colorectal cancer cells (4). Recent studies suggest that hBUBR1 is an important component of the spindle checkpoint signaling pathway, and that it targets the anaphase-promoting complex (APC) through interaction with p55CDC/hCdc2O. Specifically, whereas hBUBR1 is expressed as a 120-kDa entity in various cell lines during interphase, nocodazole (Noc)-arrested mitotic cells contain a distinct hBUBR1 species that exhibits a reduced mobility on SDS-polyacrylamide gels. Phosphatase treatment restored the mobility of the mitotic hBUBR1 species to that characteristic of the interphase state, indicating that the decrease in mobility of hBUBR1 is due to phosphorylation. Yeast two-hybrid, glutathione S-transferase pull-down, and immunoprecipitation analyses revealed that p55CDC interacts with hBUBR1. hBUBR1 also phosphorylated p55CDC in vitro. Furthermore, the APC components CDC16 and CDC27 present in lysates of Noc-treated cells interact with hBUBR1 affinity resins. Finally, we have shown that the stress-activated protein kinase p38 interacts with hBUBR1 and that Noc-induced phosphorylation of hBUBR1 in HeLa cells is blocked by pretreatment with a specific inhibitor of p38. On the basis of the known biochemical and biological activities of hBUBR1, we therefore hypothesize that activation of hBUBR1 by p38 induces its interaction with p55CDC and subsequent inhibition of the APC during spindle checkpoint activation, and that a loss of hBUBR1 function due to structural abnormalities may result in aneuploidy and cancer. To test this hypothesis, we will (i) investigate whether a dominant negative mutant of mouse BUBR1 promotes tumorigenesis in transgenic mice; (ii) determine whether p38 is an immediate upstream activator of hBUBR1 during spindle checkpoint activation; (iii) define the domain of p55CDC that interacts with hBUBR1; (iv) examine whether hBUBR1 phosphorylates p5SCDC in vivo and whether hBUBR1 and the CDK1-cyclin B complex phosphorylate p55CDC on different sites; and (v) evaluate whether hBUBR1 inhibits APC activity by phosphorylating CDC16 and CDC27. The long-term goal of this project is to determine the mechanism by which protein phosphorylation regulates the spindle checkpoint and how deregulation of it may result in cancer.